The invention relates generally to diagnostic and detection devices, and specifically to such devices and associated methods which utilize an analyte-specific luminescent signal wherein the spatial position of the luminescent signal is related to the analyte concentration.
Bioluminescence is an enzyme dependent chemical oxidation process which results in photon emission. (1, 2). The photoproteins involved in such a processxe2x80x94for example, the luciferase series of oxidative enzymesxe2x80x94are now readily available in inexpensive form, produced by recombinant means. (3). Chemiluminescence is also a chemical oxidation process resulting in photon emission, generally without the need for enzymes. (4). Adenosine triphosphate (xe2x80x9cATPxe2x80x9d) and nicotinamide adenine dinucleotide (xe2x80x9cNADHxe2x80x9d) are two molecules that play unique and central roles in biology. NADH is an ubiquitous electron donor as is the similar molecule NAD(P)H. ATP is generally recognized as one of the key energy currencies in bioenergetics. These two molecules act in a cyclic manner and can be regenerated or xe2x80x9crechargedxe2x80x9d. (5). They are the basic coupling agents of cellular metabolism. Many biochemical enzyme processes involve one of these two molecules.
Evolution has produced bioluminescence processes based on these two molecules. Examples include firefly bioluminescence, wherein firefly luciferase acts on firefly luciferin in the presence of ATP and oxygen to produce an oxidized product which chemiluminesces with a very high efficiency, and bacterial bioluminescence, wherein bacterial luciferase and an oxidoreductase, in the presence of alkyl aldehydes such as decanal, together with NADH and oxygen, produces an excited-state product which chemiluminesces. Both reactions require the presence of oxygen, although the particular luciferases and luciferins involved differ.
Literature exists describing the development of biosensors for ATP and ATP-dependent processes and for NADH and NADH-dependent processes, using the firefly and bacterial luciferase enzymes, respectively. Such biosensors generally involve fiberoptic or other waveguided means of delivering the luminescence to a sensitive device such as a photo-multiplier tube which can accurately measure light intensities. (6-9). Although one of the most portable and most sensitive photon detectors available is the human eye, it cannot be relied upon to accurately measure even relative light intensity. (10).
Luciferases have also been used as labels for a wide range of clinical diagnostic chemical tests. Since the firefly luciferase reaction is dependent on an ATP co-factor, it has been extensively used in the development of biosensors for the measurement of ATP, including detection of bacteria and other microorganisms based on the release of intracellular ATP. Instrument-based commercial biosensor kits for ATP quantification can currently attain a detection threshold of about 10xe2x88x9213 grams of ATP. (11). Likewise, bacterial luciferase which utilizes NADH, a ubiquitous chemical in bioenergetic pathways, has also been widely used for biosensing applications. Such applications have, however, been frustrated by the relative instability of these enzymes and the difficulty of incorporating them into practical and reliable biosensors.
Numerous enzymes exist which are linked to ATP consumption or ATP production, most of which are specific to another chemical substrate, for example, glycerol, glucose, etc. These xe2x80x9cfront endxe2x80x9d enzymes, coupled to a basic ATP detection device approach using luciferase and luciferin, permit the development of individual sensors or sensor channels for each of those substrates or analytes. (7-9, 12, 13). Analogous analytical systems exist for bacterial luciferase. (7,8).
The invention includes analytical chemistry systems, specific for particular analytes, including enzymes, which are specific, quantitative, rapid, direct-reading (via luminescence), sensitive, have a long shelf-life, are stable, disposable, and relatively inexpensive. The entire analytical instrument may be built into the sampling device to produce an analytical chemistry device that does not require separate instrumentation. Both analyte detection and readout utilize light produced by bioluminescence or chemiluminescence. Specificity is provided by the incorporation of specific enzymes into the device. The invention is particularly useful for the direct analysis of ATP using firefly luciferase coupled with other enzyme-mediated biochemical processes which either produce or consume ATP. As used herein, a xe2x80x9cconsumasexe2x80x9d is an enzyme which consumes the ATP, NADPH, or NADH. A xe2x80x9csynthasexe2x80x9d is an enzyme which produces ATP or NADH. However, other analytes can be linked to the ATP reaction. In a similar fashion, the invention is also applicable to analysis of analytes linked to reactions involving NADH and NAD(P)H.
A sensor according to the invention generates an analyte-specific luminescent signal in a specific spatial position that can be read by a user. It is specifically configured to produce light in a particular area of a two- or three-dimensional device, although one-dimensional geometries can be used (e.g. capillaries, rods, waveguides, etc.). It is the spatial position of the light on (or in) the device, rather than its intensity, which is related to the concentration of analyte or substrate. The human eye is much more capable of directly detecting the spatial position of a light source in a reproducible and quantitative manner, than it is of detecting changes in light intensity.
The invention produces a spatial distribution of the emitted light where such spatial distribution is proportional to the analyte concentration. The human eye or other means for light detection, e.g. chemical means such as photographic film or electronic means such as a charge coupled device (xe2x80x9cCCDxe2x80x9d), is used primarily to assess spatial position rather than absolute light intensity. This is achieved by carefully controlling the luciferase concentration and by modulating, tuning, and xe2x80x9cfilteringxe2x80x9d the ATP or NADH concentration such as through the use of a second ATP or NADH consuming enzyme. This ATP or NADH xe2x80x9cconsumasexe2x80x9d is selected to have a rapid turn over, and to serve as an ATP or NADH concentration regulator or xe2x80x9cfilterxe2x80x9d. The consumase is deposited in different spatial regions of the one-, two- or three-dimensional sensor at different concentrations.
According to the invention, a device for the quantitative analysis of one or more chemicals employs a spatial distribution of luminescence, and includes (a) a mixture of two or more chemicals that combine to produce luminescence, (b) a xe2x80x9cchemical filterxe2x80x9d or gradient that produces a spatially distributed precursor component of the mixture which results in a spatial distribution of luminescent signal, and (c) means for containing the chemicals in various spatial relationships to facilitate chemical reactions and luminescent signal detection.
The invention has industrial applicability in the areas of biology, biochemistry, biophysics, medicine, health, sports, environment, education, food, dairy, brewery, research, chemical, and related areas with chemical monitoring and analysis needs.